Oxysterols are a class of endogenous cellular lipids derived from cholesterol that have been implicated in the pathophysiology of atherosclerosis, inborn errors of metabolism, inflammation and cancer. In many cases, the proteins and molecular pathways through which these enigmatic molecules exert their powerful biological effects remain unknown. We established collaboration between a synthetic organic chemist and a cell biologist to understand how oxysterols activate the Hedgehog (Hh) signaling system, a pathway that plays important roles in development, regeneration and cancer. We discovered that a specific oxysterol, 20(S)-OHC, is an allosteric activator of the 7-pass transmembrane protein Smoothened (Smo), a human oncoprotein and key drug target in oncology. This finding significantly expands the regulatory scope of oxysterols as signaling molecules, demonstrating their capacity to function as direct agonists for both a human on co-protein and a signaling receptor. Based on preliminary work, we hypothesize that endogenous 20(S)-OHC functions as a second-messenger in Hh signaling. Using a combination of mutagenesis, photo affinity labeling, and mass spectrometry, we will map the region of Smo that interacts with 20(S)-OHC to provide a biochemical portrait of this novel class of receptor-ligand interaction (Aim 1). Using quantitative mass spectrometry and a click chemistry-based imaging assay, we will ask if Hedgehog signaling can alter cellular levels or distribution of 20(S)-OHC (Aim 2). Finally, we will develop and characterize novel Hh pathway inhibitors that are inspired by oxysterol scaffolds (Aim 3). We expect three major outcomes to emerge from the successful completion of this project: (1) An answer to the question of how Smo is regulated in cells, perhaps the longest-standing mystery in the Hh pathway, (2) a biochemical understanding of how oxysterols engage and regulate 7-pass signaling receptors, and (3) the development of an integrative toolkit that can be deployed to dissect any other oxysterol- regulated cellular process. To accomplish these goals, we have recruited a team of investigators with complementary expertise in cell biology, protein biochemistry, synthetic chemistry, and mass spectrometry.